Michael L. Nonet
Department of Anatomy & Neurobiology
6600 South Euclid Avenue - Box 8108
St. Louis, MO 63110
1995 Searle Scholar
Analysis of synapse formation and function in C. elegans.
The primary means by which most neurons communicate with their target cells is through the regulated release of neurotransmitter from synapses. Modulation of the release properties of these synapses has long been postulated, and in certain circumstances been directly demonstrated, to be a critical component of the cellular mechanisms that are thought to underlie learning and memory. Although a large number of proteins which may play significant roles in the release process have been molecularly characterized, the mechanisms that coordinate calcium-mediated fusion of transmitter-filled vesicles at the presynaptic terminal remain poorly understood. One focus of our research effort is to characterize the role of synaptic components in the release process through the analysis of C. elegans mutants that lack these molecules. Even more of a mystery are the molecular mechanisms that underlie the decisions to form synapses. Utilizing the insights and tools developed while examining functional components of the nerve terminal in the nematode, we are also using genetic approaches identify molecules that regulate the process of synaptogenesis.
Analysis of mutants with synaptic transmission defects in C. elegans in a number of laboratories including my own has led to the identification of over two dozen genes which are involved in the process. We have been characterizing mutants in genes that encode homologs of vertebrate synaptic components including the calcium binding protein synaptotagmin (snt-1), the synaptic vesicle proteins synaptobrevin (snb-1) and rab3 (rab-3) and the plasma membrane protein syntaxin (unc-64). Analysis of these mutants in C. elegans has provided significant insight into the role of these proteins in transmitter release. For example, characterization of C. elegans synaptotagmin mutants provided evidence that additional calcium sensors remain unidentified at the nerve terminal, and insight into its role in regulating endocytosis of vesicular membrane. Our present efforts are concentrated on identifying novel genes which regulate aspects of neurotransmitter release using a combination of genetic approaches (selection for resistance to pharmacological agents, supressor analysis and reverse genetics). aex-3 represents one example of a gene we identified which appears to regulate some aspect synaptic function. We isolated aex-3 mutants based on resistance to the acetylcholinesterase inhibitor aldicarb and Jim Thomas (U. of Washington) independently isolated them based on a defect in defecation behavior. We have shown that aex-3 mutants mislocalize the synaptic vesicle-associated GTP-binding protein RAB-3, but not other synaptic vesicle proteins (synaptobrevin and synaptotagmin) suggesting that AEX-3 regulates RAB-3 function. Using cellular, molecular and genetic methods, our goals are to identify and characterize novel synaptic regulators like aex-3 and determine their roles in the transmitter release pathway in the nematode. It is our expectation that homologs of these molecules will participate similarly in regulating transmitter release in vertebrates.
Other members of the lab are focusing on identifying and molecularly characterizing genes required for synaptic development by isolating mutants that fail to develop proper synapses. Recently, we have succeeded in visualizing presynaptic terminals in vivo. We have created transgenic nematodes that express a protein fusion of the synaptic vesicle membrane protein VAMP and green fluorescent protein (GFP) which localizes specifically to vesicles in vivo. When expressed under promoters that direct expression only in subsets of neurons we are able to visualize different subsets of neuron-neuron synapses and neuromuscular synapses. As GFP can be visualized directly under epi-fluorescence and the nematode is clear, we are now able to examine synaptic terminals in live animals and examine the process of synaptic development in real time. This powerful new assay enables us to isolate and examine mutants that eliminate or alter the structural features of presynaptic terminals. Such a collection of mutants will constitute the foundation for a molecular genetic analysis of the developmental events that regulate synaptogenesis.
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